Reactivation of fetal globin expression in adult erythroid cells is a prized goal to ameliorate hemoglobinopathies such as Sickle Cell Disease and beta-thalassemia. We examined the role of the Ldb1/GATA-1/TAL1/LMO2 complex, which is required for long range interaction between the adult beta-globin gene and LCR in mouse erythroid cells, in mediating beta-globin and gamma-globin expression in human cells. We investigated human NLI (Ldb1 homologue) complex occupancy and chromatin conformation of the beta-globin locus in human erythroid CD34+ cells. In addition to the LCR, we find robust NLI complex occupancy at a site downstream of the A-gamma-globin gene, within sequences of BGL3, an intergenic RNA transcript. In cells primarily transcribing beta-globin, BGL3 is not transcribed and BGL3 sequences are occupied by NLI core complex members together with co-repressor ETO2 and by gamma-globin repressor BCL11A. The LCR and beta-globin gene establish proximity in these cells. In contrast, when gamma-globin transcription is re-activated in these cells, ETO2 participation in the NLI complex at BGL3 is diminished, as is BCL11A occupancy, and both BGL3 and gamma-globin are transcribed. In these cells, proximity between the BGL3/gamma-globin region and the LCR is established. Our experiments, thus, show that alternative NLI complexes mediate gamma-globin transcription or silencing through long range LCR interactions involving an intergenic site of non-coding RNA transcription and ETO2 is critical to this process. ETO2 joins gamma-globin repressor BCL11A as a therapeutic target to ameliorate Sickle Cell Disease and beta-thalassemia. How individual globin genes establish stage specific enhancer communication is unknown and most studies have been performed in a non-chromosomal environment. We are using homologous recombination in mouse ES cells to address this issue. We targeted the mouse embryonic epsilon y and an intergenic region upstream of beta-major and then used recombinase mediated cassette exchange to insert the chicken HS4 insulator in these two positions: (1) between the LCR and the globin genes and (2) between the embryonic and adult genes. We differentiated ES cells with erythropoietin along erythroid lines and then monitored globin gene expression. The cHS4 insertion between the LCR and downstream genes reduced transcription of the embryonic ey, beta-h1 and beta major genes and resulted in non-genic transcript accumulation in the insulator. Currently, we are investigating localization of PolII and other factors such as GATA1 and Ldb1 from the LCR to 3HS1 using chromatin immunoprecipitation to understand the molecular mechanism underlying insulation. In addition, we plan to monitor globin gene transcription of ES cells that have cHS4 between the embryonic globin genes and the beta major gene. For further investigation, we are trying to derive transgenic mice which have cHS4 inserted at these two positions in the beta-globin locus. These experiments are novel since they alter transcription factor recruitment and chromatin organization in a normal chromosomal setting and will provide information on how the LCR and globin genes communicate in vivo.